The long-term objective of my lab is to obtain mechanistic insights into the role of aneuploidy in tumorogenesis. Our current focus on studying the key mechanical and regulatory events that specify accurate chromosome segregation in human cells is of direct importance towards understanding mechanisms that cause cancer and for the development of novel approaches to kill cancer cells. We have focused on identifying and characterizing the molecular components of the kinetochore as a majo[ effort towards understanding the molecular requirements for accurate chromosome segregation. The goals of this proposal are to examine how some of these proteins work together to make a functional kinetochore that links kinetochore:microtubule interactions to the mitotic checkpoint pathway. We will use molecular, biochemical and microscopic approaches to accomplish our goals. This proposal will focus on the evolutionary conserved checkpoint kinases, hBUB 1 and MPS 1, and the Cdc27 subunit ot the Anaphase Promoting Complex (APC). We will examine the mechanism by which hBUB 1 specifies the assembly of subdomain of the kinetochore that consists of checkpoint proteins hBUBR1, MAD1, MAD2 and Cdc20. These studies will be critical for understanding the dynamic nature of the interactions between checkpoint proteins and kinetochores. We will characterize the role of the hMPS 1 kinase in the mitotic checkpoint pathway by examining its importance at kinetochores and in transducing the signal from unattached kinetochores to the APC. Lastly, we have made the novel discovery that the Cdc27 subunit of the APC facilitates checkpoint inhibition of the APC in both humans and budding yeast. Our analysis of how Cdc27 accomplishes this provides a unique "bottoms- up" approach to study the signaling pathway that allows cells with even a single unattached chromosome from prematurely exiting mitosis. PERFORMANCE KEY PERSONNEL. Start with Principal Name SITE(S) (organization, city, state) Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, PA See instructions. Use continuation pages as neededto provide the required information Investigator. List all other key personnel in alphabetical order, last name first. Organization in the format shown below. Role on Project Yen, Timothy J. Institute for Cancer Research, Fox Chase Cancer Center Guacci, Vincent A. Institute for Cancer Research, Fox Chase Cancer Center Li, Yueh-chun Institute for Cancer Research, Fox Chase Cancer Center Marcus, Adam Institute for Cancer Research, Fox Chase Cancer Center Melloy, Patricia Institute for Cancer Research, Fox Chase Cancer Center Principal Investigator Co-Investigator Postdoctoral Associate Postdoctoral Associate Postdoctoral Associate Disclosure Permission Statement ApOlicable to SBIPJSTTR Only. See instructions, r3 Yes [] No PHS 398 (Rev. 05/01) Page ___ Number pages consecutively at the bottom throughout the application. Do not use suffixes such as 3a, 3b, Form Page 2 2 Principal Investigator/Program Director (Last, first,middle): YEN, Timothy L The nameof theprincipalinvestigatorprogradmirectormustbeprovidedat thetop ofeachprintedpageandeachcontinuationpage. RESEARCH GRANT TABLE OF CONTENTS Page Numbe_ 1 Face Page ....................................................................................................................................................................................... 2 Description,